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From the Departments of 1 Ophthalmology and 2 Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York.
PURPOSE. In nonconfluent cultures, TGF-ß induces differentiation of corneal fibroblasts to myofibroblasts. However, in confluent cultures, few fibroblasts differentiate to myofibroblasts after TGF-ß1 addition. This study investigated the hypothesis that functional TGF-ß receptor expression is greater in low-density cultures and is decreased in confluent cultures.
METHODS. Northern and western blot analyses were used to detect smooth muscle
(SM) 
-actin message and protein. 125I-labeled TGF-ß1
was used in a radioreceptor-binding assay as an index of functional
receptors on the cell surface of rabbit corneal fibroblast cultures
prepared either at high density (cell–cell contact) or low density
(absence of contact). Cell lysates were analyzed by SDS-PAGE and
autoradiography. Total TGF-ß receptor expression was evaluated in
western blot analysis. Smad2, a downstream effector of TGF-ß receptor
activation, was immunodetected.
RESULTS. Low-density cultures expressed more SM -actin mRNA and protein than
high-density cultures, indicating that the low-density cells were
differentiating into myofibroblasts. When 125I-TGF-ß1 was
added to low- and high-density fibroblasts, fibroblasts plated at low
density bound more than fibroblasts in high density, confluent
cultures. Furthermore, after the cells differentiated into
myofibroblasts, they continued to bind 125I-TGF-ß1.
Specificity of 125I-TGF-ß1 binding was demonstrated by
complete inhibition by excess nonradioactive TGF-ß1. Localization of
Smad2 was correlated with SM -actin induction: Smad was nuclear in
low-density cells and cytoplasmic in high-density cells. After TGF-ß1
treatment, Smad2 remained cytoplasmic in high-density cells but was
localized to nuclei in cells that were nonconfluent.
CONCLUSIONS. Low cell density is correlated with increased functional expression of TGF-ß receptors and promotion of signal transmission from these receptors. Thus, conditions that decrease cell density such as wounding favor myofibroblast differentiation in response to TGF-ß.
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